P658: DEVELOPMENT OF THE HIGHLY ACCURATE AND SENSITIVE METHOD USING THE CHIP-BASED DIGITAL PCR (LOAA) FOR THE DETECTION OF BCR::ABL1 TRANSCRIPTS DURING WHOLE PERIOD OF CHRONIC MYELOID LEUKEMIA TREATMENT
Authors: Hyun-Woo Song, Sung-Hyun Kim, Young Rok Do, Kyung-MI Ki, Soo Hyun Kim, Min-Sik Song, Seung-Shick Shin, Dong-Wook Kim
Journal: HemaSphere (2023)

Background: With various BCR::ABL1 tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) has become a manageable disease and current therapeutic goal is treatment free remission (TFR). To achieve a successful TFR in more patients, a precise assessment of minimal residual disease (MRD) is one of the prerequisite requirements. Since undetectable BCR::ABL1 fusion transcripts by conventional Taqman™-based quantitative polymerase chain reaction (qPCR) is not an indicator for complete eradication of CML clone, more sensitive technologies should be developed. Currently, to overcome some limitations of conventional qPCR, such as rigorous standardization process and various sensitivity between laboratories, digital quantitative-PCR (dqPCR) methods have been developed for accurate and precise detection and its use in the routine clinical practice is gradually expanding. However, current dqPCR methods may have limitations due to end-point interpretation and a narrow dynamic range. Therefore, we have developed a novel technology, High Dynamic Range Digital Real-time PCR (HDR-Dr. PCR™) to achieve wider dynamic range, which allows more stable, accurate and sensitive monitoring of BCR::ABL1 in a timely manner. Aims: Validation of high sensitive HDR-Dr. PCR™ technology to detect BCR::ABL1 transcripts was performed using cell lines and primary CML specimens. Methods: For linearity assessment, we used BCR::ABL1 positive (e14a2) K562 cell line diluted to 7 points by 10-fold serial dilutions from 25 to 2.5x10-5 %IS with BCR::ABL1 negative HL60 cells. With the results from cell lines, we also evaluated equivalence HDR-Dr. PCR™ and conventional qPCR using 49 clinical specimens. Based on the results, correlation analysis between qPCR and HDR-Dr. PCR™ was performed using correlation graphs and Blend-Altman plots. Results: Linearity was assessed by the dilution of RNA of K562 with RNA of HL60 using 10-fold serial dilution of up to - 7 log. The result showed accurate linearity (R2 = 0.9972) at the range of BCR::ABL1/GUSB ratio of 101 to 10-5(%). Notably, HDR-Dr. PCR™ technology allowed to detect the level of BCR::ABL1 transcript of MR 6.34 (0.00005 %IS) (Figure 1). Correlation analysis for 49 clinical samples with 0.0032 to 100 %IS showed that measured values from both qPCR and HDR-Dr. PCR™ were closely correlated with the R2 of 0.982. LOA95 (limit of agreement at 95% confidence interval) analyzed by Bland-Altman plot was -0.515 ~ 0.246 (mean = -0.134), indicating the two methods are closely correlated. Total 10 clinical specimens of CML patients with BCR::ABL1 levels of lower than 0.0032%IS was measured using both qPCR kit and HDR-Dr. PCR™ kit. The result showed that HDR-Dr. PCR™ kit detected trace amount of BCR::ABL1 transcript in additional 4 specimens compared to the detection by qPCR kit. Results with more specimens from CML patients and additional information will be updated at EHA presentation. Summary/Conclusion: HDR-Dr. PCR™ allowed to measure BCR::ABL1 transcript level of MR 6.34 with 1-step RT-dqPCR reaction in one cartridge. Comparing to conventional qRT-PCR technology, our novel digital technology may minimize false positive results and maximize the analytical sensitivity of detecting BCR::ABL1 fusion transcripts and thus, would be useful for the accurate assessment of treatment in routine clinical practice and applying TFR with wider dynamic range.Keywords: BCR::ABL, Chronic myeloid leukemia, Minimal residual disease (MRD)